2001/2002This thesis mainly direct to a few transporter metalloproteins, in particular those containing metallo-organic prosthetic groups (the iron porphyrin and the cobalt corrin complexes ). The three-dimensional structures of the native cytochrome c2 from Rhodopseudomonas palustris and of its ammonia complex have been obtained at pH 4.4 and pH 8.5, respectively. The structure of the native form has been refined in the oxidised state at 1.70 À and in the reduced state at 1.95 À resolution. These are the first highresolution crystal structures in both oxidation states of a cytochrome c2 with relatively high redox potential (+365 mV). The differences between the two oxidation states of the native form, including the position of internal water molecules, are small. The positional change of a buried water molecule, located at the heme binding pocket near to the iron bonded Met, is the most prominent structural difference observed with the change of the iron oxidation state in the c-type cytochrome structures. In all oxidised forms this water molecule is found in a position different from that found in the reduced forms. On the contrary, in the oxidised form of the cytochrome c2 from Rhodopseudomonas palustris, this water molecule is detected in a position close to that found in the reduced form. The buried water molecule plays an important role in adjusting the midpoint redox potential of c-type cytochromes with alteration of the surrounding hydrogen bond network. The unusual six-residue insertion Gly82-Ala87, which precedes the heme binding Met93, forms an isolated 310-helix secondary structural element not previously observed in other c-type cytochromes. Furthermore, this cytochrome shows an extemal methionine residue involved in a strained folding near the exposed edge of the heme. The structural comparison of the present cytochrome c2 with other c-type cytochromes has revealed that the presence of such a residue, with torsion angles and 'V of about -140 and -130°, respectively, is a typical feature of this family of proteins. The refined crystal structure of the ammonia complex, obtained at 1.15 À resolution, shows that the sulphur atom of the Met93 axial ligand does not coordinate the heme iron-atom, but is replaced by an exogenous ammonia molecule. This is the only example so far reported of an X-ray structure with the heme iron coordinated by an ammonia molecule. The detachment of Met93 is accompanied by a very localised change in the backbone conformation, involving mainly the residues Lys92, Met93 and Thr94. Previous studies under typical denaturing conditions, including high pH values and the presence of exogenous ligands, have showed that the detachment of the Met axial ligand is a basic step in the folding/unfolding process of c-type cytochromes. The ammonia-adduct represents a structural model for this important step of the unfolding pathway. Factors proposed to be important for the methionine dissociation are the strength of the H-bond between the Met93 and Tyr66 residues that stabilises the nati ve form, and the presence, in this bacterial cytochrome c2 of the rare six-residue insertion in the helix 310 conformation that increases the Met loop flexibility. Transcobalamin is a cobalamin binding protein in mammalian plasma that facilitates the cellular uptake of vitamin B 12 • Whereas the X-ray structures of severa! B12-enzymes are available, no structural information on B12-transporting proteins has so far been reported. Human and bovine transcobalamin were expressed using ricombinant yeast cells and purified. Human transcobalamin was successfully crystallised usmg polyethylene glycol and ethanol as precipitants. Crystals belong to the orthorhombic space group P212121, with unit-cell parameters a= 49.04, b = 145.27, and e= 164.96 À. A complete data set to 3.2 À resolution was collected from a single crystal using s ynchrotron radiati on (Elettra, Trieste). Estimati on of the crystal packing (V m = 3.2 À 3 Da-1) and self-rotation function analysis suggest the presence of two molecules in the asymmetric unit related by a pseudo-twofold symmetry. Bovine transcobalamin was crystallised using polyethylene glycol and MPD. Crystals belong to the monoclinic space group P21 (a = 95.32, b = 100.19, e = 98.73 À and ~ = 96.9 À), and diffract to better then 2.0 À resolution. MAD data sets were collected at Elettra (Trieste) from a single crystal of the bovine protein, using the cobalamin Co atom as anomalous scatterer. Attempts to find the position of Co atoms in the monoclinic cell from Bijvoet difference Patterson maps are under way.XV Ciclo1973Versione digitalizzata della tesi di dottorato cartacea
